Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

Localizing the RPGR protein along the cilium: a new method to determine efficacies to treat RPGR mutations.

Retinal dystrophies constitute a group of clinically and genetically heterogeneous diseases that cause visual impairment. As treatments are not readily available, readout assays performed in patient-derived cells can aid in the development and comparative analysis of therapeutic approaches. We describe a new method with which the localization of the retinitis pigmentosa GTPase regulator (RPGR) protein along the cilium can be used as a measure for treatment efficacy. In a patient-derived fibroblast cell line, we found that the RPGR protein is mislocalized along the ciliary axoneme. The patient carried a point mutation that leads to skipping of RPGR exon 10. We confirmed that this skipping is causative for the impaired localization of RPGR using a U7 small nuclear RNA (U7snRNA)-based antisense approach in control cells. Treatment of the patient-derived fibroblasts with therapeutic U1snRNA significantly corrected the proteins' mislocalization. In this proof of principle study, we show that detecting the RPGR protein along the cilium provides a reliable and quantifiable readout assay to evaluate the efficacy of therapies intended to correct or silence RPGR gene mutations. This method opens the possibility to compare different therapeutic agents, and thus facilitate the identification of treatment options for the clinically and molecularly complex RPGR-associated diseases.

Semi-quantitative chemical analysis of hard coatings by Raman micro-spectroscopy: the aluminium chromium nitride system as an example.

A new method for chemical analyses of nitride-based hard coatings is presented. Raman band shifts in the spectra of Al(x)Cr(1-x)N coatings, deposited by physical vapour deposition from Al(x)Cr(1-x) targets with x (T,Al) = 0, 0.25, 0.50, 0.70 and 0.85, are calibrated using compositional data of the coatings derived by elastic recoil detection analysis (ERDA) and electron probe micro-analysis (EPMA). Inserting the composition-dependent Raman shift of a combinatorial acoustic-optic lattice mode into an empirically derived equation allows the determination of Al/Cr ratios of the coating with an accuracy of about +/-2%. Spot, line and area analyses of coated cemented carbide and cold work steel samples by using a computer-controlled, motorized x,y-stage are demonstrated and the most important errors influencing precision and accuracy are discussed. Figure Raman map of a coated cold-work steel sample.

[Viral and nonviral gene therapy for treatment of retinal diseases]. / Virale und nichtvirale Gentherapieansätze zur Behandlung von Netzhauterkrankungen.

The development of gene therapeutic approaches offers new perspectives for the treatment of retinal diseases. The use of both, nonviral methods employing oligonucleotides as well as viral expression vectors provide the possibility to treat neovascularization defects and retinal degeneration, respectively. The mechanism by which the therapeutic oligonucleotides (antisense oligonucleotides, aptamers and siRNA) work is based on degradation of specific transcripts. Consequently, a reduction of the corresponding protein, which is involved in the particular pathogenesis, follows. In contrast, viral vector transduction can substitute the disease-associated gene with an intact copy. So far, animal models have successfully contributed to the development of gene therapeutic medication and further treatments are at the recruiting phase of clinical trials.

Imaging of fullerene-like structures in CNx thin films by electron microscopy; sample preparation artefacts due to ion-beam milling.

The microstructure of CN(x) thin films, deposited by reactive magnetron sputtering, was investigated by transmission electron microscopy (TEM) at 200kV in plan-view and cross-sectional samples. Imaging artefacts arise in high-resolution TEM due to overlap of nm-sized fullerene-like features for specimen thickness above 5nm. The thinnest and apparently artefact-free areas were obtained at the fracture edges of plan-view specimens floated-off from NaCl substrates. Cross-sectional samples were prepared by ion-beam milling at low energy to minimize sample preparation artefacts. The depth of the ion-bombardment-induced surface amorphization was determined by TEM cross sections of ion-milled fullerene-like CN(x) surfaces. The thickness of the damaged surface layer at 5 degrees grazing incidence was 13 and 10nm at 3 and 0.8keV, respectively, which is approximately three times larger than that observed on Si prepared under the same conditions. The shallowest damage depth, observed for 0.25keV, was less than 1nm. Chemical changes due to N loss and graphitization were also observed by X-ray photoelectron spectroscopy. As a consequence of chemical effects, sputtering rates of CN(x) films were similar to that of Si, which enables relatively fast ion-milling procedure compared to carbon compounds. No electron beam damage of fullerene-like CN(x) was observed at 200kV.

Cross-linked nano-onions of carbon nitride in the solid phase: existence of a novel C(48)N(12) aza-fullerene.

We report a new fullerenelike material consisting of cross-linked nano-onions of C and N. Growth of the onion shells takes place atom by atom on a substrate surface and yields thin solid films during magnetron sputter deposition. Electron microscopy and energy loss spectroscopy show that the core shell contains up to 20 at. % N corresponding to C(48)N(12) aza-fullerene composition. Nanoindentation of this nanostructured material gives high resilience with hardness 7 GPa, Young's modulus 37 GPa, and complete elastic recovery after loading with 0.5 mN to a depth of 75 nm. Total energy calculations show the stability of C(60-2n)N(2n) aza-fullerenes and suggest the existence of a novel C(48)N(12) molecule.

A new case of congenital cyst of the pancreas.

A new case of congenital cyst of the pancreas is reported. A 34-year-old woman was admitted with a painless, large, epigastric mass. Ultrasonography revealed an anechogenic retrogastric tumor. Computed tomography scan described a liquid cyst of the pancreas which was 15 cm in diameter. During surgery, a well-delimited translucent cyst was found and no local malignancy was observed. Extensive distal pancreatic resection with preservation of the spleen was performed and a thin part of cephalic pancreas was preserved. The liquid of the cyst did not contain any mucus. Microscopic study of the cyst wall described normal cuboidal cells and congenital cyst of the pancreas was diagnosed. Several diagnoses including hydatid cyst, pseudocyst and cystic tumors of the pancreas are discussed. Before surgery, lack of acute pancreatitis in recent medical history rules out pseudocyst and hydatid cyst. During the operation, if cystadenocarcinoma is easily ruled out, macrocystic serous cystadenoma is more difficult to exclude. Only histological examination of the cystic wall confirms the difference between cystadenoma and congenital cyst which remains an exceptional entity.

Review and evaluation of anatomy sites on the Internet (updated 1999).

In a preceding study, we had reviewed and evaluated the anatomy sites available on the Internet. The evolution of the available sites, the disappearance of certain sites and the appearance of new sites led us to update this list. In addition, the German-speaking sites were included in the study, which previously included only the Anglophone and French-speaking sites. Forty-eight sites were indexed and their addresses are available on the site of the Laboratory of Anatomy of the Faculty of Medicine Lyon-Nord (http://rockefeller.univ-lyon1.fr/Anatomie-Lyon- Nord). Compared to the scores allotted in 1998, we noted in 1999 a significant increase (p = 0.03) in the total score (12.13 +/- 2.98 vs. 11.23 +/- 2.28/20), which shows that the quality of the anatomic sites available on the Internet has improved.

French mirror site of the NPAC visible human viewer: first year evaluation.

The NPAC visible human viewer (NPAC VHV), graphical interface written in JAVA, freely accessible by the Web, allows the display of anatomic cross-sections of the Visible Human Project developed by the National Library of Medicine. In April 1997, the Medical Media Library of Lyons undertook the construction of a French-language mirror site of the NPAC VHV. The aim of this work is to evaluate first year utilisation of this site. From May 1st, 1997 to April 30th, 1998, the mirror site was consulted 34,752 times. In 45.14% of cases, the request came from France, in 4.42% of cases from Belgium, in 3.98% from Canada and in 2.12% from Switzerland. Other connections came either from a country responsible for fewer than 1% of connections or from unidentified computers. Data analysis showed a peak of connections between 15:00 and 17:00, and an increased number of connections from September to March 1998. The NPAC VHV is housed in 5 sites in the world. It is a software very simple to use. As the figures have no legends, it is more appropriate for group teaching than for self-teaching.

Prospecting and evaluation of the anatomy sites on the Internet.

The Internet undoubtedly has an important part to play in medical teaching, generally speaking, and particularly in anatomy. We therefore undertook to survey, list, explore and study the various sites written in French or English devoted to anatomy on the Internet in order to evaluate them. Sites were identified from their URL address, by search engine or by hypertext links found in already listed sites. Useless and non-relevant sites were excluded. Fifty-two sites were selected and evaluated as to their navigability, illustrations, text and general presentation. Addresses of theses sites are available on a web page (http:/(/)www.rockefeller.univ-lyon1.fr/Anato mie-Lyon-Nord; section liens, section Galerie Virtuelle). This directory could be a very useful working tool for the use of teachers and students. It will be regularly updated.

Gastrocolic vein. Definition and report of two cases of avulsion.

The gastrocolic v. or Henle's gastrocolic trunk was described in 1868 [9]. We suggest defining this vein as the confluence of the right gastroepiploic and right upper colic vv. We report two original cases of avulsion of the gastrocolic v. occurring during a blunt abdominal trauma. The aim of this paper is a description, based on the literature, of the anatomy of the gastrocolic v. in order to precise the lesional mechanism. The gastrocolic v. is present in 70% of individuals. It is short (less than 25 mm) but of major calibre (3 to 10 mm). The gastrocolic v. is situated close beneath the root of the transverse mesocolon, and travels along the anterior surface of the head of the pancreas. Anatomic variations are detailed and a meta-analysis of interpretable studies was made. Both the supra- and infra-mesocolic surgical approaches are described. The radiologic and surgical importance of the gastrocolic v. is discussed. The lesional mechanism in both our cases of injury of the gastrocolic v. is explained.

[Resistance of the the coraco-clavicular ligaments to traction: clinical application]. / Etude de la résistance à la traction des ligaments coraco-claviculaires: applications cliniques.

One method for treating chronic incapacitating acromioclavicular dislocation is to resect the external extremity of the clavicle and to stabilise the stump by ligamentoplastic procedures, using the coracoclavicular ligament. The purpose of this work was to evaluate the mechanical quality of the ligamentoplastic approach. Twelve fresh cadavers, average age 80 years, were studied. The samples taken were 24 coracoclavicular ligaments, 24 coraco-acromial ligaments, 9 tendons from the palmaris longus muscle and 9 iliotibial tracts. The ligaments removed were tested in a Instron traction machine at a speed of 10 cm/mn. The mechanical properties of the coracoclavicular and coraco-acromial ligaments were studied. For comparison, those of the tendon of the palmaris longus muscle and the iliotibial tract were also studied. The results show the pre-rupture resistance of the coraco-acromial ligament to be 50% lower than that of the trapezoid and conoid parts of the coracoclavicular ligaments taken together. These results suggest the validity of ligamentoplastic treatment using the coraco-acromial ligament, but that reinforcement, using a tendon from the palmaris longus muscle or a piece of the iliotibial tract, may also be necessary, especially for subjects taking part in sports or with well-developed musculature.

[Appendectomy: evaluation of medical record maintenance in a series of 200 cases]. / Appendicectomies: évaluation de la tenue du dossier médical sur une série de 200 cas.

To improve the management of appendicular syndromes, two hundred files of patients undergoing appendectomy in an emergency surgery department between January 1993 and August 1994 were submitted to a retrospective and descriptive study with evaluation of the medical file content. To evaluate clinical and investigations data collecting, a histopathological review protocol was elaborated to obtain an objective and reliable criterion of the degree of inflammation of the appendix. This review was possible only for 197 files that were included. Data collecting rates are inferior than expected rates, particularly for the association of temperature, abdominal defence and white blood cells count that was found in only 159 files (80.7%). Data were less collected for women, patients operated by celioscopy and when histopathologic review concluded to absence of acute inflammation. Rate of acute appendicitis was 73.6% (145/197). Appendectomies without acute inflammation were higher for women (34.6% = 37/107) than for men (16.7% = 15/90) and for patients who underwent celioscopic appendectomy (42.9% = 33/77) than for patients who underwent Mac Burney appendectomy (16% = 19/119). These results highlight the need to improve competition of medical files with better collection of clinical data, which could lead to improve care quality and management of operated appendicular syndromes, first step to a reduction of the number of appendectomies. A global assessment of file completion could be proposed on the basis of clinical audit.

[Diaphragm rupture in closed trauma]. / Rupture du diaphragme dans les traumatismes fermés.

Ruptures of the diaphram occur in approximately 2% of cases of severe thoraco-abdominal trauma. They are present on the left in 80% of cases. The rupture is cupolar, sagittal or transversal, with peripheral desinsertion (the last always observed on the right), or paravertebral and retropericardiac posterior tears. Movement of the abdominal viscera toward the thorax can be progressive, with signs appearing only after 3 or 4 days or more. Any attempt to evacuate an intrathoracic effusion should be made carefully. In half the cases in polytraumatic patients, the lesion is confirmed by clinical suspicion or by appropriate surgical exploration. The first emergency step is repair. In cases of recent occurrence and for reasons of abdominal safety, coeliotomy is preferred. Mortality ranges from 20 to 30% and depends on the polytraumatic state but also on heart and respiratory failure and on infectious complications.

[Preliminary and multidisciplinary study of three Coptic mummies of the Testut-Latarjet museum at Lyon]. / Etude pluridisciplinaire et préliminaire de trois momies coptes du Musée Testut-Latarjet de Lyon.

In 1901 Albert Gayet raked up from Antinoe three mummies exhibited to day at the Anatomy Museum of Lyon. The study of the three mummies was made in detail as to their dress, anthropometric and scannographic findings. The clothes were characteristic of coptic civilization. The radiographic date gave a life span of around 40 years. The X-ray imagery shows the remains of cerebral and visceral organs. The sexual criteria are thought to be those of two women and undetermined for the child. Later, several investigations like endoscopic autopsy, tooth microscopy and chromosomic map will be necessary.

Imagery rehearsal treatment of chronic nightmares: with a thirty month follow-up.

Nightmare frequency and self-rated distress were assessed retrospectively in two groups of chronic nightmare sufferers 30 months after treatment. In the initial phase, the image group (N = 9) learned a cognitive-behavioral technique (imagery rehearsal) for the treatment of nightmares. They were taught in one group session to: (1) record a nightmare; (2) change it (usually to something positive); and (3) rehearse the new images daily. The record group (N = 10) recorded nightmares during the first month only and learned imagery rehearsal subsequent to 3-month follow-up measurements. At 3 months and at 30 months, both groups had significantly fewer nightmares, but only the rehearsal group had less total distress. The results support the theory that nightmares are a primary sleep disorder rather than a symptom of an underlying psychiatric problem.

Dose changes in long- and medium-term intrathecal morphine therapy of cancer pain.

Intrathecal morphine analgesia for the treatment of cancer pain was administered using implanted ports and drug delivery systems (DDS) in 79 patients. Effective control of the pain was achieved in nearly all patients; in only two patients was the use of the DDS discarded because of relative ineffectiveness. Fifty-three manual drug release systems (41 lumbar, 12 ventricular) and 26 lumbar ports were used. Forty patients survived more than 2 months; the maximum survival time was 560 days (mean survival time, 80 days with a port system, 100 days with a manual DDS). Patients still alive at the time of this study, i.e., with unknown survival time, were excluded. The initial mean daily dose was 8.5 mg in lumbar ports, 2.75 mg in lumbar DDS, and 0.2 mg with intraventricular application. Dose change patterns disclosed no alteration of the initial dose in 18 of 26 port patients, an initial increase in 4, a preterminal increase in 3, and a single intermittent increase in 1 patient. Of 40 lumbar DDS patients, 13 showed a constant dose, 9 an initial, 3 a preterminal, and 5 an intermittent increase. Three patients with less than 2 months' survival time had a rather continuous increase. All long-time survivors (i.e., with more than 2 months' survival time) reached a plateau and remained there until a preterminal if any increase occurred. These findings suggest the morphine dosage to be indicative of the progress of the disease rather than of a drug tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)